Rho family GTPases are critical in regulating cancer metastasis and cell motility. Of particular importance, Cdc42 has been shown to directly affect metastatic migration of primary tumors via signaling through a sub- class of Wiskott Aldrich Syndrome protein neural-WASP (N-WASP) to initiate invadopodial protrusions exhibiting potent matrix degrading capacity. Here, we will develop a high-throughput, homogenous assay that reports the guanine nucleotide state of Cdc42 GTPase, and use it to monitor Cdc42 - N-WASP interaction and to screen against small molecule inhibitors that can selectively attenuate Cdc42 - N-WASP interaction but not WASP interaction which is important in hematopoietic cells. The assay will make use of novel solvatochromic dyes that are extraordinarily bright (extinction coefficient >180,000, QY >0.9) providing sensitive assay with an excellent z'-factor. The dyes will be attached to a protein domain that binds only to the activated form of the Cdc42 protein. Activation of Cdc42 will lead to binding of the domain, with consequent changes in the fluorescence of the dye. We will develop homogenous, "mix and measure" HTS assays using all purified components. Secondary screens will identify inhibitors specific for Cdc42 - N-WASP interaction but not WASP interaction, and those specific over the other Cdc42 isoforms including TC-10 and TCL. These assays will enable us to study the unique role played by Cdc42 in metastasis and invasion, potentially identify inhibitors of a new target for anti-metastasis therapy and demonstrate the value of the new dyes for development of other sensitive homogeneous assays. PUBLIC HEALTH RELEVANCE: This project will identify novel therapeutic targets for aggressive cancers by screening for new inhibitors of Cdc42 - N-WASP interaction, a critical component of formation of invasive protrusion structures during cancer metastasis. Cdc42 regulates formation of invadopodia via specific interaction with its downstream effector N-WASP, controlling the cancer migration through extracellular matrix layers. By designing a state of the art biosensor that can detect Cdc42 - N-WASP interaction at an unprecedented sensitivity and accuracy, we will be able to design a highly robust high-throughput assay reagent targeting this important protein.